Group I introns are fascinating RNA molecules whose molecular architecture mediates accurate and efficient RNA splicing reactions. This project directly addresses three central issues regarding ribozymes and RNA splicing- First, how does the molecular structure of ribozymes result in catalytic activity and site-specificity? Second, what are the specific contributions of protein and RNA to catalytic ribonucleoprotein (RNP) complexes? Third, can ribozymes be developed into highly selective tools for inactivation and repair of targeted RNA molecules in mammalian cells? The development of ribozymes catalyzing-trans 5' splice site reactions has led to an explosion of information on the first step of group I splicing Insights into 3' splice site reactions have lagged because constructs for catalysis of trans 3' splice site reactions were unavailable. Progress to date includes: (i) Establishing the structural framework for 3' splice site reactions, (ii) Development and preliminary utilization of two fundamentally different ribozymes that catalyze 3' splice site reactions in trans, and (iii) Development of novel and powerful in vitro selection techniques for the analysis of 3' splice site reactions. The Specific Aims of this project are to: (1) Optimize and characterize two novel group I ribozymes that catalyze 3' splice site reactions in trans. (2) Define the tertiary structure responsible for forward and reverse 3' splice site reactions. (3) Identify structural distinctions between self-splicing and protein-dependent group I introns. (4) Explore potential applications of group I 3' splice site ribozymes. Results will be of immediate importance to those studying splicing, ribozyme catalysis, molecular recognition, RNA structure, RNA-protein interactions, and molecular evolution. In addition, trans-acting ribozymes emerging from this project may be used for targeted RNA inactivation and repair in vivo, with important implications for basic science and gene-specific molecular therapeutics.